dTTP Incorporation Research Articles

Replication of the Aristolochic Acid I Adenine Adduct (ALI-N6-A) by a Model Translesion Synthesis DNA Polymerase: Structural Insights on the Induction of Transversion Mutations from Molecular Dynamics Simulations.

Exposure to aristolochic acid I and II (AAI and AAII) has been implicated in aristolochic acid nephropathy and urothelial carcinoma. The toxicological effects of AAs are attributed to their ability to form aristolacatam (AL)-purine DNA adducts. Among these lesions, the AL-adenine (ALI-N6-A and ALII-N6-A) adducts cause the "signature" A → T transversion mutations associated with AA genotoxicity. To provide the currently missing structural basis for the induction of these signature mutations, the present work uses classical all-atom molecular dynamics simulations to examine different (i.e., preinsertion, insertion, and postextension) stages of replication past the most abundant AA adduct (ALI-N6-A) by a representative lesion-bypass DNA polymerase (Dpo4). Our analysis reveals that, before dNTP incorporation (i.e., preinsertion step), ALI-N6-A adopts a nearly planar conformation at the N6-linkage and the ALI moiety intercalates within the DNA helix. Since this conformation occupies the dNTP binding site, the same planar lesion conformation results in a significant distortion of the polymerase active site at the insertion step and therefore replication will likely not be successful. However, if ALI-N6-A undergoes a small conformational change to introduce non-planarity at the N6-linkage during the insertion step, minimal distortion occurs in the Dpo4 active site upon incorporation of dATP. This insertion and subsequent extension would initially lead to A:A mismatches and then result in A → T transversion mutations during the second round of replication. In contrast, if a large conformation flip of the ALI moiety occurs at the insertion step to reorient the bulky moiety from an intercalated position into the major groove, dTTP (non-mutagenic) incorporation will be favored. Molecular dynamics (MD) simulations on postextension complexes reveal that damaged DNA will likely further rearrange during later replication steps to acquire a base-displaced intercalated conformation that is similar to that previously reported for (unbound) ALI-N6-A adducted DNA, with the exception of slight non-planarity at the lesion site. Overall, our results provide a structural explanation for both the successful non-mutagenic lesion bypass and the preferential misincorporation of dATP opposite ALI-N6-A and thereby rationalize the previously reported induction of A → T signature transversion mutations associated with AAs. This work should thereby inspire future biochemical experiments and modeling studies on the replication of this important class of DNA lesions by related human translesion synthesis polymerases.

Epigenetically modified N6-methyladenine inhibits DNA replication by human DNA polymerase iota

Among human four Y-family DNA polymerases, hPol ι is exceptionally error-prone in DNA synthesis. 6 mA plays significant roles in epigenetic regulation of numerous biological processes. Nonetheless, its effects on DNA replication by hPol ι is still unclear. In this work, we found that 6 mA and Hyp, the intermediate of 6 mA, inhibited the replication of DNA by hPol ι. 6 mA lost priority in extension beyond 6 mA:T pair, partially reducing dTTP incorporation efficiency and inhibiting next-base extension. Hyp was prone to dCTP incorporation and extension beyond Hyp:C instead of Hyp:T pair. Statistically, 6 mA primarily reduced the burst incorporation rate (kpol) and slightly increased the dissociation constant (Kd,dTTP). However, Hyp mainly increased the Kd,dCTP yet did not affect the kpol, both reducing the burst incorporation efficiency (kpol/Kd,dCTP). 6 mA together with Hyp weakened the binding affinity of hPol ι to DNA in binary or ternary complex. The misincorporation opposite 6 mA or Hyp further weakened this binding affinity. The methyl group in 6 mA doesn’t almost affect the H-bond formation with dTTP, therefore mildly inhibiting dTTP incorporation. As an analogue of G, Hyp can form only two H-bonds with dCTP, thus reducing dCTP incorporation. This work provides a new insight in how the epigenetically modified 6 mA and its intermediate Hyp affect replication of DNA by human DNA polymerase ι.

Epigenetic DNA modification N6-methyladenine inhibits DNA replication by Sulfolobus solfataricus Y-family DNA polymerase Dpo4

Dpo4 is a representative model of Y-family DNA polymerase and is therefore one of the most intensively studied DNA polymerase. 6 mA, an epigenetic marker, plays important roles in regulation of various biological processes. However, its effects on DNA replication by Dpo4 is completely unknown. Here, we found that 6 mA and its intermediate Hyp inhibits primer extension by Dpo4, showing an obvious blockage just one nucleotide before 6 mA or Hyp. 6 mA reduces dTTP incorporation efficiency, next-base extension efficiency, binding affinity of DNA to Dpo4, binding affinity of dTTP to Dpo4-DNA complex, the fraction of productive Dpo4 or productive ternary complex, and the burst incorporation rate, explaining the inhibition effects of 6 mA on DNA replication by Dpo4. Hyp is similar to G and dCTP is preferentially incorporated opposite Hyp by Dpo4, resulting in A:T to G:C mutation. Relative to dTTP incorporation opposite unmodified A, Hyp reduces dCTP incorporation efficiency, next-base extension efficiency, the priority in extension beyond correct pair, binding affinity of Dpo4 to DNA, binding of dCTP to Dpo4-DNA complex, and the burst incorporation efficiency, explaining the inhibition effects of Hyp on DNA replication by Dpo4. This work provides insight in the effects of epigenetically modified 6 mA and Hyp on DNA replication by a representative Y-family DNA polymerase Dpo4.

Psoromic Acid, a Lichen-Derived Molecule, Inhibits the Replication of HSV-1 and HSV-2, and Inactivates HSV-1 DNA Polymerase: Shedding Light on Antiherpetic Properties.

Psoromic acid (PA), a bioactive lichen-derived compound, was investigated for its inhibitory properties against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), along with the inhibitory effect on HSV-1 DNA polymerase, which is a key enzyme that plays an essential role in HSV-1 replication cycle. PA was found to notably inhibit HSV-1 replication (50% inhibitory concentration (IC50): 1.9 μM; selectivity index (SI): 163.2) compared with the standard drug acyclovir (ACV) (IC50: 2.6 μM; SI: 119.2). The combination of PA with ACV has led to potent inhibitory activity against HSV-1 replication (IC50: 1.1 µM; SI: 281.8) compared with that of ACV. Moreover, PA displayed equivalent inhibitory action against HSV-2 replication (50% effective concentration (EC50): 2.7 μM; SI: 114.8) compared with that of ACV (EC50: 2.8 μM; SI: 110.7). The inhibition potency of PA in combination with ACV against HSV-2 replication was also detected (EC50: 1.8 µM; SI: 172.2). Further, PA was observed to effectively inhibit HSV-1 DNA polymerase (as a non-nucleoside inhibitor) with respect to dTTP incorporation in a competitive inhibition mode (half maximal inhibitory concentration (IC50): 0.7 μM; inhibition constant (Ki): 0.3 μM) compared with reference drugs aphidicolin (IC50: 0.8 μM; Ki: 0.4 μM) and ACV triphosphate (ACV-TP) (IC50: 0.9 μM; Ki: 0.5 μM). It is noteworthy that the mechanism by which PA-induced anti-HSV-1 activity was related to its inhibitory action against HSV-1 DNA polymerase. Furthermore, the outcomes of in vitro experiments were authenticated using molecular docking analyses, as the molecular interactions of PA with the active sites of HSV-1 DNA polymerase and HSV-2 protease (an essential enzyme required for HSV-2 replication) were revealed. Since this is a first report on the above-mentioned properties, we can conclude that PA might be a future drug for the treatment of HSV infections as well as a promising lead molecule for further anti-HSV drug design.

Epigenetic DNA Modification N6-Methyladenine Inhibits DNA Replication by DNA Polymerase of Pseudomonas aeruginosa Phage PaP1.

N6-methyladenine (6mA), a newly identified epigenetic modification, plays important roles in regulation of various biological processes. However, the effect of 6mA on DNA replication has been little addressed. In this work, we investigated how 6mA affected DNA replication by DNA polymerase of Pseudomonas aeruginosa Phage PaP1 (gp90 exo-). The presence of 6mA, as well as its intermediate hypoxanthine (Hyp), inhibited DNA replication by gp90 exo-. The 6mA reduced dTTP incorporation efficiency by 10-fold and inhibited next-base extension efficiency by 100-fold. Differently, dCTP was preferentially incorporated opposite Hyp among four dNTPs. Gp90 exo- reduced the extension priority beyond the 6mA:T pair rather than the 6mA:C mispair and preferred to extend beyond Hyp:C rather than the Hyp:T pair. Incorporation of dTTP opposite 6mA and dCTP opposite Hyp showed fast burst phases. The burst rate and burst amplitude were both reduced for 6mA compared with unmodified A. Moreover, the total incorporation efficiency ( kpol/ Kd,dNTP) was decreased for dTTP incorporation opposite 6mA and dCTP incorporation opposite Hyp compared with dTTP incorporation opposite A. 6mA reduced the incorporation rate ( kpol), and Hyp increased the dissociation constant ( Kd,dNTP). However, 6mA or Hyp on template did not affect the binding of DNA polymerase to DNA in binary or ternary complexes. This work provides new insight into the inhibited effects of epigenetic modification of 6mA on DNA replication in PaP1.

Error-prone bypass of O6-methylguanine by DNA polymerase of Pseudomonas aeruginosa phage PaP1

O6-Methylguanine (O6-MeG) is highly mutagenic and is commonly found in DNA exposed to methylating agents, generally leads to G:C to A:T mutagenesis. To study DNA replication encountering O6-MeG by the DNA polymerase (gp90) of P. aeruginosa phage PaP1, we analyzed steady-state and pre-steady-state kinetics of nucleotide incorporation opposite O6-MeG by gp90 exo−. O6-MeG partially inhibited full-length extension by gp90 exo−. O6-MeG greatly reduces dNTP incorporation efficiency, resulting in 67-fold preferential error-prone incorporation of dTTP than dCTP. Gp90 exo− extends beyond T:O6-MeG 2-fold more efficiently than C:O6-MeG. Incorporation of dCTP opposite G and incorporation of dCTP or dTTP opposite O6-MeG show fast burst phases. The pre-steady-state incorporation efficiency (kpol/Kd,dNTP) is decreased in the order of dCTP:G>dTTP:O6-MeG>dCTP:O6-MeG. The presence of O6-MeG at template does not affect the binding affinity of polymerase to DNA but it weakened their binding in the presence of dCTP and Mg2+. Misincorporation of dTTP opposite O6-MeG further weakens the binding affinity of polymerase to DNA. The priority of dTTP incorporation opposite O6-MeG is originated from the fact that dTTP can induce a faster conformational change step and a faster chemical step than dCTP. This study reveals that gp90 bypasses O6-MeG in an error-prone manner and provides further understanding in DNA replication encountering mutagenic alkylation DNA damage for P. aeruginosa phage PaP1.

Kinetic Mechanism of DNA Polymerization Catalyzed by Human DNA Polymerase ε

Eukaryotes require highly accurate and processive DNA polymerases to ensure faithful and efficient replication of their genomes. DNA polymerase ε (Polε) has been shown to catalyze leading-strand DNA synthesis during replication in vivo, but little is known about the kinetic mechanism of polymerization catalyzed by this replicative enzyme. To elucidate this mechanism, we have generated a truncated, exonuclease-deficient mutant of the catalytic subunit of human Polε (Polε exo-) and carried out pre-steady-state kinetic analysis of this enzyme. Our results show that Polε exo-, as other DNA polymerases, follows an induced-fit mechanism when catalyzing correct nucleotide incorporation. Polε exo- binds DNA with a Kd(DNA) of 79 nM and dissociates from the E·DNA binary complex with a rate constant of 0.021 s(-1). Although Polε exo- binds a correct incoming nucleotide weakly with a Kd(dTTP) of 31 μM, it catalyzes correct nucleotide incorporation at a fast rate constant of 248 s(-1) at 20 °C. Both a large reaction amplitude difference (42%) between pulse-chase and pulse-quench assays and a small elemental effect (0.9) for correct dTTP incorporation suggest that a slow conformational change preceding the chemistry step limits the rate of correct nucleotide incorporation. In addition, our kinetic analysis shows that Polε exo- exhibits low processivity during polymerization. To catalyze leading-strand synthesis in vivo, Polε likely interacts with its three smaller subunits and additional replication factors in order to assemble a replication complex and significantly enhance its polymerization processivity.

Incorporation of Nucleoside Probes Opposite O6‐Methylguanine by Sulfolobus solfataricus DNA Polymerase Dpo4: Importance of Hydrogen Bonding

O⁶-Methylguanine (O⁶-MeG) is a mutagenic DNA lesion, arising from the action of methylating agents on guanine (G) in DNA. Dpo4, an archaeal low-fidelity Y-family DNA polymerase involved in translesion DNA synthesis (TLS), is a model for studying how human Y-family polymerases bypass DNA adducts. Previous work showed that Dpo4-mediated dTTP incorporation is favored opposite O⁶-MeG rather than opposite G. However, factors influencing the preference of Dpo4 to incorporate dTTP opposite O⁶-MeG are not fully defined. In this study, we investigated the influence of structural features of incoming dNTPs on their enzymatic incorporation opposite O⁶-MeG in a DNA template. To this end, we utilized a new fluorescence-based primer extension assay to evaluate the incorporation efficiency of a panel of synthetic dNTPs opposite G or O⁶-MeG by Dpo4. In single-dNTP primer extension studies, the synthetic dNTPs were preferentially incorporated opposite G, relative to O⁶-MeG. Moreover, pyrimidine-based dNTPs were generally better incorporated than purine-based syn-conformation dNTPs. The results suggest that hydrophobicity of the incoming dNTP appears to have little influence on the process of nucleotide selection by Dpo4, with hydrogen bonding capacity being a major influence. Additionally, modifications at the C2-position of dCTP increase the selectivity for incorporation opposite O⁶-MeG without a significant loss of efficiency.

Replication past the Butadiene Diepoxide-Derived DNA Adduct S-[4-(N6-Deoxyadenosinyl)-2,3-dihydroxybutyl]glutathione by DNA Polymerases

1,2,3,4-Diepoxybutane (DEB), a metabolite of the carcinogen butadiene, has been shown to cause glutathione (GSH)-dependent base substitution mutations, especially A:T to G:C mutations in Salmonella typhimurium TA1535 [Cho, S. H., et al. (2010) Chem. Res. Toxicol. 23, 1544] and Escherichia coli TRG8 cells [Cho, S. H., and Guengerich, F. P. (2012) Chem. Res. Toxicol. 25, 1522]. We previously identified S-[4-(N(6)-deoxyadenosinyl)-2,3-dihydroxybutyl]GSH [N(6)dA-(OH)2butyl-GSH] as a major adduct in the reaction of S-(2-hydroxy-3,4-epoxybutyl)glutathione (DEB-GSH conjugate) with nucleosides and calf thymus DNA and in vivo in livers of mice and rats treated with DEB [Cho, S. H., and Guengerich, F. P. (2012) Chem. Res. Toxicol. 25, 706]. For investigation of the miscoding potential of the major DEB-GSH conjugate-derived DNA adduct [N(6)dA-(OH)2butyl-GSH] and the effect of GSH conjugation on replication of DEB, extension studies were performed in duplex DNA substrates containing the site-specifically incorporated N(6)dA-(OH)2butyl-GSH adduct, N(6)-(2,3,4-trihydroxybutyl)deoxyadenosine adduct (N(6)dA-butanetriol), or unmodified deoxyadenosine (dA) by human DNA polymerases (Pol) η, ι, and κ, bacteriophage polymerase T7, and Sulfolobus solfataricus polymerase Dpo4. Although dTTP incorporation was the most preferred addition opposite the N(6)dA-(OH)2butyl-GSH adduct, N(6)dA-butanetriol adduct, or unmodified dA for all polymerases, the dCTP misincorporation frequency opposite N(6)dA-(OH)2butyl-GSH was significantly higher than that opposite the N(6)dA-butanetriol adduct or unmodified dA with Pol κ or Pol T7. LC-MS/MS analysis of full-length primer extension products confirmed that Pol κ or Pol T7 incorporated the incorrect base C opposite the N(6)dA-(OH)2butyl-GSH lesion. These results indicate the relevance of GSH-containing adducts for the A:T to G:C mutations produced by DEB.

Electronic Measurements of Single-Molecule Processing by DNA Polymerase I (Klenow Fragment)

Bioconjugating single molecules of the Klenow fragment of DNA polymerase I into electronic nanocircuits allowed electrical recordings of enzymatic function and dynamic variability with the resolution of individual nucleotide incorporation events. Continuous recordings of DNA polymerase processing multiple homopolymeric DNA templates extended over 600 s and through >10,000 bond-forming events. An enzymatic processivity of 42 nucleotides for a template of the same length was directly observed. Statistical analysis determined key kinetic parameters for the enzyme's open and closed conformations. Consistent with these nanocircuit-based observations, the enzyme's closed complex forms a phosphodiester bond in a highly efficient process >99.8% of the time, with a mean duration of only 0.3 ms for all four dNTPs. The rate-limiting step for catalysis occurs during the enzyme's open state, but with a nearly 2-fold longer duration for dATP or dTTP incorporation than for dCTP or dGTP into complementary, homopolymeric DNA templates. Taken together, the results provide a wealth of new information complementing prior work on the mechanism and dynamics of DNA polymerase I.

Structural Basis for Proficient Incorporation of dTTP Opposite O6-Methylguanine by Human DNA Polymerase ι

O(6)-methylguanine (O(6)-methylG) is highly mutagenic and is commonly found in DNA exposed to methylating agents, even physiological ones (e.g. S-adenosylmethionine). The efficiency of a truncated, catalytic DNA polymerase ι core enzyme was determined for nucleoside triphosphate incorporation opposite O(6)-methylG, using steady-state kinetic analyses. The results presented here corroborate previous work from this laboratory using full-length pol ι, which showed that dTTP incorporation occurs with high efficiency opposite O(6)-methylG. Misincorporation of dTTP opposite O(6)-methylG occurred with ∼6-fold higher efficiency than incorporation of dCTP. Crystal structures of the truncated form of pol ι with O(6)-methylG as the template base and incoming dCTP or dTTP were solved and showed that O(6)-methylG is rotated into the syn conformation in the pol ι active site and that dTTP misincorporation by pol ι is the result of Hoogsteen base pairing with the adduct. Both dCTP and dTTP base paired with the Hoogsteen edge of O(6)-methylG. A single, short hydrogen bond formed between the N3 atom of dTTP and the N7 atom of O(6)-methylG. Protonation of the N3 atom of dCTP and bifurcation of the N3 hydrogen between the N7 and O(6) atoms of O(6)-methylG allow base pairing of the lesion with dCTP. We conclude that differences in the Hoogsteen hydrogen bonding between nucleotides is the main factor in the preferential selectivity of dTTP opposite O(6)-methylG by human pol ι, in contrast to the mispairing modes observed previously for O(6)-methylG in the structures of the model DNA polymerases Sulfolobus solfataricus Dpo4 and Bacillus stearothermophilus DNA polymerase I.

Characterization of HIV-1 Enzyme Reverse Transcriptase Inhibition by the Compound 6-Chloro-1,4-Dihydro-4-Oxo-1-(β-D-Ribofuranosyl) Quinoline-3-Carboxylic Acid Through Kinetic and In Silico Studies

We recently described that the chloroxoquinolinic ribonucleoside 6-chloro-1,4-dihydro-4-oxo-1-(beta-D-ribofuranosyl) quinoline-3-carboxylic acid (compound A) inhibits the human immunodeficiency virus type 1 (HIV-1) enzyme reverse transcriptase (RT), and its replication in primary cells. Based on these findings, we performed kinetic studies to investigate the mode of inhibition of compound A and its aglycan analog (compound B). We found that both molecules inhibited RT activity independently of the template/primer used. Nevertheless, compound A was 10-fold more potent than compound B. Compound A inhibited the RNA-dependent DNA polymerase (RDDP) activity of RT with an uncompetitive and a noncompetitive mode of action with respect to dTTP incorporation and to template/primer (TP) uptake, respectively. The kinetic pattern of the inhibition displayed by compound A was probably due to its greater affinity for the ternary complex (RT-TP-dNTP) than the enzyme alone or the binary complex (RT-TP). Besides, by means of molecular modeling, we show that compound A bound on the NNRTI binding pocket of RT. However, our molecule targets such a site by making novel interactions with the enzyme RT, when compared to NNRTIs. These include a hydrogen bridge between the 2'-OH of our compound and the Tyr675 of the enzyme RT's chain B. Therefore, compound A is able to synergize with both a NRTI (AZT-TP) and a NNRTI (efavirenz). Taken together, our results suggest that compound A displays a novel mechanism of action, which may be different from classical NRTIs and NNRTIs.

R964C Mutation of DNA Polymerase γ Imparts Increased Stavudine Toxicity by Decreasing Nucleoside Analog Discrimination and Impairing Polymerase Activity

The R964C mutation of human DNA polymerase gamma was recently linked to stavudine (d4T)-mediated mitochondrial toxicity. We utilized pre-steady-state kinetics to determine the effect of this mutation on incorporation of natural substrate dTTP and the active metabolite of d4T (d4TTP). The R964C polymerase gamma holoenzyme demonstrated a 33% decrease in dTTP incorporation efficiency and a threefold-lower d4TTP discrimination relative to that of the wild-type polymerase gamma, providing a mechanistic basis for genetic predisposition to nucleoside reverse transcriptase inhibitor toxicity.

Efficient and Erroneous Incorporation of Oxidized DNA Precursors by Human DNA Polymerase η

Altered oxidative metabolism is a property of many tumor cells. Oxidation of DNA precursors, i.e., dNTP pool, as well as DNA is a major source of mutagenesis and carcinogenesis. Here, we report the remarkable nature of human DNA polymerase eta that incorporates oxidized dNTPs into a nascent DNA strand in an efficient and erroneous manner. The polymerase almost exclusively incorporated 8-hydroxy-dGTP (8-OH-dGTP) opposite template adenine (A) at 60% efficiency of normal dTTP incorporation, and incorporated 2-hydroxy-dATP (2-OH-dATP) opposite template thymine (T), guanine (G), or cytosine (C) at substantial rates. The synthetic primers having 8-hydroxy-G paired with template A or 2-hydroxy-A paired with template T, G, or C at the termini were efficiently extended. In contrast, human DNA polymerase iota incorporated 8-OH-dGTP opposite template A with much lower efficiency and did not incorporate 2-OH-dATP opposite any of the template bases. It did not extend the primers having the oxidized bases at the termini either. We propose that human DNA polymerase eta may participate in oxidative mutagenesis through the efficient and erroneous incorporation of oxidized dNTPs during DNA synthesis.

Sulfolobus solfataricus DNA Polymerase Dpo4 Is Partially Inhibited by “Wobble” Pairing between O6-Methylguanine and Cytosine, but Accurate Bypass Is Preferred

We examined the effect of a single O6-methylguanine (O6-MeG) template residue on catalysis by a model Y family polymerase, Dpo4 from Sulfolobus solfataricus. Mass spectral analysis of Dpo4-catalyzed extension products revealed that the enzyme accurately bypasses O6-MeG, with C being the major product (approximately 70%) and T or A being the minor species (approximately 20% or approximately 10%, respectively), consistent with steady-state kinetic parameters. Transient-state kinetic experiments revealed that kpol, the maximum forward rate constant describing polymerization, for dCTP incorporation opposite O6-MeG was approximately 6-fold slower than observed for unmodified G, and no measurable product was observed for dTTP incorporation in the pre-steady state. The lack of any structural information regarding how O6-MeG paired in a polymerase active site led us to perform x-ray crystallographic studies, which show that "wobble" pairing occurs between C and O6-MeG. A structure containing T opposite O6-MeG was solved, but much of the ribose and pyrimidine base density was disordered, in accordance with a much higher Km,dTTP that drives the difference in efficiency between C and T incorporation. The more stabilized C:O6-MeG pairing reinforces the importance of hydrogen bonding with respect to nucleotide selection within a geometrically tolerant polymerase active site.

Kinetic Effect of a Downstream Strand and Its 5′-Terminal Moieties on Single Nucleotide Gap-filling Synthesis Catalyzed by Human DNA Polymerase λ

During short-patch base excision repair, the excision of a 5'-terminal 2-deoxyribose-5-phosphate moiety of the downstream strand by the 5'-2-deoxyribose-5-phosphate lyase activity of either DNA polymerase beta or lambda is believed to occur after each respective enzyme catalyzes gap-filling DNA synthesis. Yet the effects of this 5'-terminal 2-deoxyribose-5-phosphate moiety on the polymerase activities of these two enzymes have never been quantitatively determined. Moreover, x-ray crystal structures of truncated polymerase lambda have revealed that the downstream strand and its 5'-phosphate group of gapped DNA interact intensely with the dRPase domain, but the kinetic effect of these interactions is unclear. Here, we utilized pre-steady state kinetic methods to systematically investigate the effect of a downstream strand and its 5'-moieties on the polymerase activity of the full-length human polymerase lambda. The downstream strand and its 5'-phosphate were both found to increase nucleotide incorporation efficiency (kp/Kd) by 15 and 11-fold, respectively, with the increase procured by the effect on the nucleotide incorporation rate constant kp rather than the ground state nucleotide binding affinity Kd. With 4 single nucleotide-gapped DNA substrates containing a 1,2-dideoxyribose-5-phosphate moiety, a 2-deoxyribose-5-phosphate mimic, we measured the incorporation efficiencies of 16 possible nucleotides. Our results demonstrate that although this 5'-terminal 2-deoxyribose-5-phosphate mimic does not affect the fidelity of polymerase lambda, it moderately decreased the polymerase efficiency by 3.4-fold. Moreover, this decrease in polymerase efficiency is due to a drop of similar magnitude in kp rather than Kd. The implication of the downstream strand and its 5'-moieties on the kinetics of gap-filling synthesis is discussed.

Evidence That HIV-1 Reverse Transcriptase Employs the DNA 3′ End-directed Primary/Secondary RNase H Cleavage Mechanism during Synthesis and Strand Transfer

We previously analyzed strand transfers catalyzed by human immunodeficiency virus, type 1 reverse transcriptase (RT) in a hairpin-containing RNA template system. In this system, RT produces a series of adjacent RNase H cuts before the hairpin base on the first, or donor template that clears a region of the donor, facilitating invasion by the second, or acceptor RNA. Here we analyze characteristics of the prominent cuts before the hairpin base and their role in strand transfers. Analysis of the template cleavage pattern during synthesis suggested that the RT performs DNA 3' end-directed primary and secondary cuts while paused at the hairpin base and that these cuts contribute to creation of the invasion site. RT catalyzed similar cleavages on a substrate representing a paused cDNA-template intermediate. DNA 3' end-directed secondary cuts, which require positioning of the polymerase active site downstream of the primer terminus, had previously not been specifically identified during synthesis. Our findings indicate that during synthesis DNA 3' end-directed primary and secondary cuts occur at pause sites. RT mutants with substitutions at the His(539) residue in the RNase H active site were defective in secondary cleavages. Analysis of the template cleavage pattern generated by the His(539) mutants during synthesis revealed inefficient cleavage at the invasion site, correlating with defects in strand transfer. Overall, results indicate RT can catalyze pause-associated DNA 3' end-directed primary and secondary cuts during synthesis and these cuts can contribute to strand transfer by creation of an invasion site.

AP endonuclease deficiency results in extreme sensitivity to thymidine deprivation

Thymidine depletion is toxic to virtually all actively growing cells. The fundamental mechanism responsible for thymidineless death remains unknown. One event thought to be critical in causing the toxicity of thymidine depletion is a sharp rise in the ratio of dUTP to dTTP and subsequent incorporation of dUTP into DNA. Maneuvers to alter dUTP levels appear to alter the toxicity of thymidine depletion. However, loss of uracil-DNA-N-glycosylase activity does not appear to change the toxicity of thymidine deprivation significantly. This study proposes to define the role of uracil base excision repair (BER) in mediating thymidineless death. The toxicity of thymidine deprivation induced by the antifolate aminopterin was measured in a series of mutant Saccharomyces cerevisiae strains deficient in various steps in uracil-BER. Most mutants displayed modest changes in their sensitivity to aminopterin, with the exception of cells lacking the abasic endonuclease Apn1. apn1 mutants displayed a profound sensitivity to aminopterin that was relieved in an apn1 ung1 double mutant. Wild-type and apn1 mutants displayed similar levels of DNA damage and S-phase arrest during aminopterin treatment. A significant portion of cell killing occurred after removal of aminopterin in both wild-type and apn1 mutant cells. apn1 mutants showed a complete inability to re-initiate DNA replication following removal of aminopterin. These findings suggest recovery from arrest is a crucial step in determining the response to thymidine deprivation and that interruptions in uracil-BER increase the toxicity of thymidine deprivation by blocking re-initiation of replication rather than inciting global DNA damage. Inhibition of apurinic/apyrimidinic endonuclease may therefore be a reasonable approach to increase the efficacy of anticancer chemotherapies based on thymidine depletion.

Reiterative template switching: The effect of single-strand homopolymeric DNA on non-template-directed nucleotide addition by DNA polymerase

Template switching occurs when DNA polymerase juxtaposes two discontinuous DNA molecules with 3′-terminally complementary ends generated through non-template-directed nucleotide addition. We examined whether juxtaposition of homopolymeric single-stranded oligonucleotides affects non-templated addition. We hypothesized that if DNA polymerase first juxtaposed the two substrates, then the non-template-directed nucleotide addition of any deoxynucleotide would decrease in the presence of its non-complementary template. For dATP, product formation was unaffected by non-complementary substrates. In contrast, dCTP and dGTP incorporation decreased to varying degrees while dTTP incorporation increased in the presence of oligodeoxythymidine but decreased for other non-complementary homopolymers. Interestingly, the presence of complementary templates strongly influenced the formation of highly periodic products indicative of reiterative template switching. Transient template synapsis was observed and found to be dependent on the non-templated sequence added: 3–4 A:T or 1–2 G:C base pairs were needed for stable synapsis, suggesting that base pairing plays a more important role in the active site of the enzyme than previously thought.

DNA Elongation by the Human DNA Polymerase λ Polymerase and Terminal Transferase Activities Are Differentially Coordinated by Proliferating Cell Nuclear Antigen and Replication Protein A

DNA polymerase lambda contains template-dependent (DNA polymerase) and template-independent (terminal transferase) activities. In this study we enzymologically characterized the terminal transferase activity of polymerase lambda (pol lambda-tdt). Pol lambda-tdt activity was strongly influenced by the nature of the 3'-terminal sequence of the DNA substrate, and it required a single-stranded (ss) DNA 3'-overhang of about 9-12 nucleotides for optimal activity. The strong preference observed for pyrimidine versus purine nucleotide incorporation was found to be due, at least partially, to a steric block imposed by the residue Tyr-505 in the active site of pol lambda. Pol lambda-tdt was found to be able to elongate a 3'-ssDNA end by two alternative mechanisms: first, a template-independent one resulting in addition of 1 or 2 nucleotides, and second, a template-dependent one where a homopolymeric tract as short as 3 nucleotides at the 3'-end could be used as a template to direct DNA polymerization by a looping back mechanism. Furthermore repetitive cycles of DNA synthesis resulted in the expansion of such a short homopolymeric terminal sequence. Most importantly we found that the proliferating cell nuclear antigen was able to selectively block the looping back mechanism while stimulating the single terminal nucleotide addition. Finally replication protein A completely suppressed the transferase activity of pol lambda while stimulating the polymerase activity, suggesting that proliferating cell nuclear antigen and replication protein A can coordinate the polymerase and the terminal transferase activities of pol lambda.